› Optimized Protocol

DCT-1011 Vs. 05-2006

› for T84 Cell Line [ATCC]

page 1 of 7

Cell Line Nucleofector® Kit T for T84 Cell Line [ATCC] Cell type

Origin

Human lung metastase from colorectal carcinoma [ATCC® CCL-248™; frozen vial].

Morphology

Epithelial cell.

Example for nucleofection® of T84 cells. 100

% transfection efficiency B

A

80 60 40 20 0 24 h Average transfection efficiency of T84 cells. Cells were nucleofected with program T-05/T-005 and 2 µgof pmaxGFPTM. 24 hours post nucleofection, the cells were analyzed by flow cytometry. Cell Viability (compared to un-nucleofected control) is around 82% 24 hours postnucleofection.

Chapter

T84 cells were nucleofected using the Cell Line Nucleofector Kit T, program T-05/T-005 and 2 µg of pmaxGFP. 24 hours post nucleofection the cells were analyzed by fluorescence microscopy.

Contents

1

Procedure outline & important advice

2

Product description

3

Protocol 3.1 3.2 3.3 3.4 3.5

4 5

› › › › ›

Required reagents DNA preparation

and quality

Cell culture Important controls and vector information Nucleofection protocol

Recommended literature 5

amaxa GmbH Europe/World Scientific Support +49 (0)221-99199-400 [email protected]

› www.amaxa.com

amaxa Inc. USA Scientific Support (240) 632-9110 [email protected]

› Optimized Protocol

DCT-1011 Vs. 05-2006

› for T84 Cell Line [ATCC]

page 2 of 7

Procedure outline & important advice

1

1.

Procedure outline

Important advice

Culturing of cells before

› › › ›

nucleofection. (For details see 3.3.)

Replace medium every 2-3 days. Passage cells twice a week. Subcultivation ratio of 1:2 to 1:3. Subculture cells 3-4 days before nucleofection with a ratio of 1:2 to 1:3.

2.

Combine the cells of inte-

Contents of one nucleofection sample:

rest, DNA or siRNA and the

› 2 x 106 cells (optimal cell number) › 2 µg highly purified plasmid DNA

appropriate cell-type specific Nucleofector Solu-

(in max. 10 µl) or 0.5 - 3 µg siRNA

› 100 µl Nucleofector Solution T

tion and transfer to an

›

amaxa certified cuvette. (For details see 3.5.) Perform each sample separately to avoid storing the cells longer than 15 min in Nucleofector Solution T.

3.

Choose the cell-type speci-

› Optimal Nucleofector program: T-05*/T-005**

fic program. Insert the cuvette into the Nucleofector and press the “X” button to start the program. (For details see 3.5.)

› *for Nucleofector I Device › **for Nucleofector II Device

4.

Rinse the cuvette with culture

medium

amaxa

using

certified

an

pipette.

Transfer the cells into the

› Using an amaxa certified pipette, immediately remove sample from the cuvette with 500 µl prewarmed medium.

› Transfer directly to 37°C.

culture dish. (For details see 3.5.)

amaxa GmbH Europe/World Scientific Support +49 (0)221-99199-400 [email protected]

› www.amaxa.com

amaxa Inc. USA Scientific Support (240) 632-9110 [email protected]

› Optimized Protocol

DCT-1011 Vs. 05-2006

› for T84 Cell Line [ATCC]

2

page 3 of 7

Product description

Cat. No.

VCA-1002

Kit components

2.25 ml Cell Line Nucleofector® Solution T 0.5 ml Supplement 1 30 µg pmaxGFPTM (0.5 µg/µl in 10 mM Tris pH 8.0) 25 certified cuvettes 25 plastic pipettes

Size

25 reactions

Storage and stability

Store Nucleofector Solution, Supplement and pmaxGFP at 4°C. For long term storage pmaxGFP is ideally stored at -20°C. The expiry date is printed on the Solution Box.

3

Protocol

3.1 Medium

› Required reagents 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium containing 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES and 0.5 mM sodium pyruvate, 95% [ATCC; Cat. No. 30-2006]; fetal bovine serum, 5% [ATCC; Cat. No. 30-2020].

Trypsin

0.5 mg/ml Trypsin; 0.2 mg/ml EDTA in PBS.

Treatment

3.2

› DNA preparation and quality

The quality and the concentration of DNA used for nucleofection plays a central role for the efficiency of gene transfer. We strongly recommend the use of high quality products for plasmid purification like QIAGEN EndoFree® Plasmid Kits [Cat. No. 12391 Giga Kit, 12362 Maxi Kit, 12381 Mega Kit]. The purified DNA should be resuspended in deionized water or TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0) with a concentration between 1-5 µg/µl. Please check the purity of each plasmid preparation by measurement of the A260:A280 ratio, according to QIAGEN protocol.

amaxa GmbH Europe/World Scientific Support +49 (0)221-99199-400 [email protected]

› www.amaxa.com

amaxa Inc. USA Scientific Support (240) 632-9110 [email protected]

› Optimized Protocol

DCT-1011 Vs. 05-2006

› for T84 Cell Line [ATCC]

3.3

page 4 of 7

› Cell culture

Culture conditions Replace medium every 2-3 days. Passage interval

Cells should be passaged twice a week. A subcultivation ratio of 1:2 to 1:3 is recommended.

Seeding conditions 5 x 104 - 1 x 105 cell/cm2. Culture conditions before nucleofection

› The cells should be preferably passaged 3-4 days before nucleofection with a ratio of ratio 1:2 to 1:3.

› Use early passages for nucleofection.

Note

Contamination of cell culture with mycoplasma is a widely spread phenomenon that might negatively influence experimental results. We recommend the use of NormocinTM [Cat. No. VZA-1001], a new antibiotic formula specifically developed to protect sensitive cell lines from mycoplasma infection and microbial contaminations. For more information and ordering info see www.amaxa.com/antibiotics.

3.4 Positive control

› Important controls and vector information

We strongly recommend establishing the Nucleofector technology with the positive control vector pmaxGFPTM as provided in this kit. pmaxGFP encodes the green fluorescent protein (GFP) from copepod Pontellina p. Just like eGFP expressing cells, maxGFP expressing cells can easily be analyzed by fluorescence microscopy or flow cytometry to monitor transfection efficiency.

Esp 3l (7) Eco 31l (18) Nsil (27)

TM

Esp 3l (2667) Kpnl (980) Nhel (988) Eco47lll (993) Agel (997)

BspTl (1891) Eco31l (1896) Esp3l (1909)

amaxa GmbH Europe/World Scientific Support +49 (0)221-99199-400 [email protected]

Bglll (1676) Xhol (1680) Sacl (1687)

› www.amaxa.com

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› Optimized Protocol

DCT-1011 Vs. 05-2006

› for T84 Cell Line [ATCC]

Negative control

page 5 of 7

We recommend you always perform two control samples to assess the initial quality of cell culture and the potential influences of nucleofection or amount/purity of DNA on cell viabilty. control 1 Recommended amount of cells in Nucleofector Solution with DNA but without application of the program (alternatively: untreated cells) (Cells + Solution + DNA - program) control 2 Recommended amount of cells in Nucleofector Solution without DNA with application of the program (Cells + Solution - DNA + program)

Vector information

If using IRES sequences in your vectors, please remember that the gene encoded 3’ of the IRES sequence is usually expressed to a lesser extent than the upstream gene, and in some cell types may not be expressed at all. As alternatives we suggest either: co-transfecting two (or more) plasmids, using one plasmid with each gene under the control of its own promoter, or making a GFP fusion.

3.5 Preparation of Nucleofector Solution

› Nucleofection protocol

Add 0.5 ml Supplement to 2.25 ml Nucleofector Solution and mix gently. The Nucleofector Solution is now ready to use and is stable for 3 months at 4°C. Note the date of addition on the vial.

One nucleofection sample contains

› 2 x 106 cells › 2 µg plasmid DNA (in 1-5 µl H2O or TE) or 2 µg pmaxGFP or 0.5 - 3µg siRNA › 100 µl Nucleofector Solution T

For more details about the nucleofection of siRNA: www.amaxa.com/RNAi

Preparation of

1. Cultivate the required number of cells.

samples

2. Prepare 2 µg DNA or 0.5 - 3 µg siRNA for each sample. 3. Pre-warm the supplemented Cell Line Nucleofector Solution T to room temperature. Pre-warm an aliquot of culture medium at 37°C in a 50 ml tube (500 µl per sample). 4. Prepare 6-well plates by filling appropriate number of wells with 1 ml of culture medium containing supplements and serum. Pre-incubate plates in a humidified 37°C/5% CO2 incubator. 5. Remove the medium from the cell culture. Wash cells once with PBS.

amaxa GmbH Europe/World Scientific Support +49 (0)221-99199-400 [email protected]

› www.amaxa.com

amaxa Inc. USA Scientific Support (240) 632-9110 [email protected]

› Optimized Protocol

DCT-1011 Vs. 05-2006

› for T84 Cell Line [ATCC]

page 6 of 7

6. Harvest the cells e.g. with Trypsin/EDTA and stop the trypsinization with supplemented culture medium or PBS/0.5% BSA (see Nucleofector Manual for details), making sure to thoroughly resuspend cells. 7. Take an aliquot of trypsinized cell suspension and count the cells to determine the cell density. 8. Centrifuge the required number of cells (2 x 106 cells per nucleofection sample) at 90xg at room temperature for 10 min. Discard supernatant completely so that no residual medium covers the cell pellet. 9. Thoroughly resuspend the pellet in room temperature Cell Line Nucleofector Solution T to a final concentration of 2 x 106 cells/100 µl. It is important to obtain a single cell suspension! Avoid storing the cell suspension longer than 15 min in Nucleofector Solution as this reduces cell viability and gene transfer efficiency. Important: Steps 10-14 should be performed for each sample separately. Nucleofection

10. Mix 100 µl of cell suspension with 2 µg DNA or 0.5 - 3 µg siRNA. 11. Transfer the nucleofection sample into an amaxa certified cuvette. Make sure that the sample covers the bottom of the cuvette, avoid air bubbles while pipetting. Close the cuvette with the blue cap. 12.Select the appropriate Nucleofector program, T-05/T-005 (see Nucleofector Manual for details). Insert the cuvette into the cuvette holder (Nucleofector I : rotate carousel to final position) and press the “X” button to start the program. 13. To avoid damage to the cells remove the sample from the cuvette immediately after the program has finished (display showing "OK"). Take the cuvette out of the holder. Add 500 µl of the prewarmed culture medium and transfer the sample into the prepared 6-well plates. Alternatively, transfer the sample into a 1.5 ml microcentrifuge tube and place it in a 37°C heat block. Take the cuvette out of the holder. To transfer the cells from the cuvettes, we strongly recommend using the plastic pipettes provided in the kit to prevent damage and loss of cells. 14. Press the “X” button to reset the Nucleofector. 15. Repeat steps 10-14 for the remaining samples.

Cultivation after nucleofection

16. If you have incubated the samples in 1.5 ml microcentrifuge tubes transfer them into the prepared 6-well plates. 17. Incubate cells in a humidified 37°C/5% CO2 incubator. Following nucleofection, gene expression should be analyzed at different times. Depending on the gene, expression is often detectable after 4-8 hours. If this is not the case, the incubation period may be prolonged up to 24 hours.

amaxa GmbH Europe/World Scientific Support +49 (0)221-99199-400 [email protected]

› www.amaxa.com

amaxa Inc. USA Scientific Support (240) 632-9110 [email protected]

› Optimized Protocol

DCT-1011 Vs. 05-2006

› for T84 Cell Line [ATCC]

4

page 7 of 7

Recommended literature

For an up-to-date list of all Nucleofector references, please refer to: www.amaxa.com/citations

* amaxa’s Nucleofector® process, Nucleofector® device and Nucleofector® Solutions are covered by PCT applications PCT/EP01/07348, PCT/DE02/01489, PCT/DE02/01483 and other pending patents and domestic or foreign applications corresponding thereto. * amaxa, Nucleofector, nucleofection and maxGFP are trademarks of amaxa GmbH. * This kit contains a proprietary nucleic acid coding for a proprietary copepod fluorescent protein intended to be used as a positive control with this amaxa product only. Any use of the proprietary nucleic acid or protein other than as a positive control with this amaxa product is strictly prohibited. USE IN ANY OTHER APPLICATION REQUIRES A LICENSE FROM EVROGEN. To obtain such a license, please contact Evrogen at [email protected]. * The CMV promoter is covered under U.S. Patents 5,168,062 and 5,385,839 and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242. * QIAGEN and EndoFree are trademarks of QIAGEN. * ATCC® and the ATCC Catalog Marks are trademarks of ATCC. * All other product and company names mentioned herein are the trademarks of their respective owners.

amaxa GmbH Europe/World Scientific Support +49 (0)221-99199-400 [email protected]

› www.amaxa.com

amaxa Inc. USA Scientific Support (240) 632-9110 [email protected]